Small-molecule agonists for type-2 orexin receptor

ABSTRACT

Methods and compositions for agonizing a type-2 orexin receptor (OX2R) in a cell determined to be in need thereof, including the general method of (a) administering to a subject a cyclic guanidinyl OX2R agonist and (b) detecting a resultant enhanced wakefulness or increased resistance to diet-induced accumulation of body fat, or abbreviated recovery from general anesthesia or jet lag.

This application claims priority to U.S. Ser. No. 61/058,914, filed Jun.4, 2008, and having the same title and inventor.

The field of the invention is small-molecule agonists for type-2 orexinreceptor

Introduction

This invention describes several chemically distinct classes ofnon-peptidic, small-molecule agonists for the type-2 orexin receptors(OX2R). Orexins are hypothalamic neuropeptides that are importantlyimplicated in sleep/wake control and body weight homeostasis. Orexinproducing neurons are exclusively localized in the lateral hypothalamicarea. The peptides act on two G protein-coupled receptors termed OX1Rand OX2R. It has been demonstrated that deficiency in orexin/OX2Rsignaling causes the sleep disorder narcolepsy in humans, mice and dogs.Narcolepsy is a socially debilitating disorder characterized by aninability to properly maintain wakefulness (excessive daytimesleepiness, sleep attacks), and a pathological intrusion of signs of REMsleep into wakefulness (cataplexy, hipnagorgic hallucination, sleepparalysis, etc). Narcolepsy affects 1/1,000˜1/2,000 individuals, and isa non-progressive, life-long condition. A vast majority (>90%) of humannarcoleptics lacks detectable levels of orexin peptides in thecerebrospinal fluid due to a highly specific (probably autoimmune)degeneration of orexin neurons, indicating that human narcolepsy is an“orexin deficiency syndrome.”

A transgenic mouse strain that has been engineered to mimic theneurochemical situation in human narcoleptics, i.e., with a postnatalloss of orexin neurons, exhibits all symptoms of narcolepsy/cataplexy.It has been further demonstrated that these mice can then be effectivelytreated for narcolepsy by providing exogenous orexin either genetically(via a transgene) or pharmacologically (via intracerebroventricularinjections). Hence, orexin replacement therapy is expected to providesimilarly effectively treat human narcolepsy patients.

Orexins themselves are peptides (thus orally inactive) and blood-brainbarrier impermeable; they cannot be used as an orally active therapeuticagent. However, if we can develop an orally active, blood-brain barrierpermeable, small-molecule OX2R agonist, such compound will be useful asa drug for the treatment of narcolepsy.

This invention provides a cellular assay method that can be used forhigh-throughput screening of OX2R agonists from chemical compoundlibraries. The screen method uses transfected cell lines co-expressingthe human OX2R cDNA (or another control receptor cDNA) and anNFAT-responsive luciferase reporter. Furthermore, the invention providesresults from actual screenings utilizing this assay method, identifyingseveral distinct classes of active compounds, which can be optionallyfurther optimized chemically using the same assay.

The practical uses for OX2R agonists go beyond the treatment ofnarcolepsy. Since orexin has been established as an endogenousneurotransmitter to maintain proper wakefulness, orexin receptoragonists effect a highly “natural” form of wakefulness. Therefore, suchagonists may be used to treat medical conditions accompanying daytimesleepiness, such as nighttime insomnia, and depression with hypersomnia.

It has also been shown that orexin/OX2R signaling acts as a net-negativeregulator of body weight homeostasis. Thus, deficiencies in OX2Rsignaling can cause (besides narcolepsy) obesity in mice and humans,whereas transgenic OX2R over-activation renders mice resistant to highfat diet-induced obesity. Furthermore, intracerebroventricular infusionof a previously known peptidic OX2R-selective agonist similarly preventshigh fat diet-induced obesity in mice. Therefore, orally active OX2Ragonists also provide treatments of obesity and associated metabolicsyndrome.

Orexin deficiency causes delayed recovery from gas anesthesia in mice,which effect can be antagonized by orexin and orexin agonists. See, Kelzet al. An essential role for orexins in emergence from generalanesthesia. Proc. Natl. Acad. Sci. USA 105:1309-1314 (2008). Hence, thesubject orexin agonists may also be used to facilitate or expediterecovery from general anesthesia.

The active OX2R agonists provided in this invention also provide leadcompounds for the development of alternative and more potent forms withdesirable pharmacokinetics, bioavailability, nontoxicity, and otherproperties. Such derivative compounds may be used, inter alia and afterproper clinical trials, as therapeutic agents for treating (1)narcolepsy; (2) other neurologic conditions accompanying daytimesleepiness, such as night time insomnia and depression with hypersomnia,sleep apnea, jet lag, etc.; (3) obesity and metabolic syndrome; and (4)undesirable extension and lingering effects of general anesthesia.

The general class of OX2R agonists identified herein was independentlydescribed for use as local analgesics (see, WO 03/105779), and apreferred embodiment described herein has the same structure as compound#11 in Table 1 of WO 03/105779.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for agonizing a type-2orexin receptor (OX2R) in a cell determined to be in need thereof. Thegeneral methods comprise the steps of (a) contacting the receptor withan OX2R agonist of formula I:

as further defined in various embodiments below, or a pharmaceuticallyacceptable salt thereof, or prodrug thereof; and (b) detecting aresultant agonizing or activation of the receptor.

In particular embodiments, the method provides a drug screen oroptimization assay, wherein the cell is in vitro and further comprises atranscriptional reporter of OX2R activation, and the agonizing isdetected as increased expression of the reporter.

In particular embodiments, the method provides a therapy or prophylaxis,wherein the cell is in situ in a subject, the OX2R agonist is orallyadministered to the subject, and the agonizing or activation is inferredfrom a resultant physiological effect, such as enhanced wakefulness orincreased resistance to diet-induced accumulation of body fat, orenhanced recovery from general anesthesia or jet lag. In more specificembodiments, this in situ protocol comprises (i) administering to thesubject the OX2R agonist regularly and chronically; (ii) administeringto the subject the OX2R agonist in conjunction with an effective amountof a different compound which promotes wakefulness, anti-obesity, orrecovery from general anesthesia or jet lag; or (iii) administering tothe subject the OX2R agonist in unit dosage form, and comprising theantecedent step of removing a capsule or tablet comprising the unitdosage form from a multipack adapted for sequential use.

The invention also provides pharmaceutical compositions comprising anOX2R agonist of formula I (as defined in various embodiments below),particularly as coformulated with a different compound which promoteswakefulness, anti-obesity, or recovery from general anesthesia or jetlag, and/or formulated in unit dosages separately packaged in amultipack adapted for sequential use.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Yan 7874: Metabolism Studies

FIG. 2. Yan 7874: Pharmacokinetics Studies

FIG. 3. Yan 7874: SAR Studies around the cyclic guanidinium scaffold.

DEFINITIONS

As used herein, terms have the following meanings:

Alkyl, alkoxy, alkoxyalkyl, alkylthio: saturated acyclic moieties, withstraight or branched chains, having the indicated number of carbonatoms. Alkyl groups are hydrocarbyl moieties; alkoxy and alkoxyalkylgroups have an oxygen atom in the chain; alkylthio groups have a sulfuratom in the chain. Examples include methyl, ethyl, and the variouspropyl, butyl, pentyl, hexyl and octyl groups, methoxy, ethoxy,n-propoxy, isopropoxy, methoxymethyl, ethoxymethyl, n-propoxyethyl,methylthio, ethylthio, n-propylthio and n-butylthio.

Alkyleneoxy; alkylenedioxy: includes methylenedioxy, —OCH₂O—,ethylenedioxy, —OCH₂CH₂O— and ethyleneoxy, —CH₂CH₂O—.

Cycloalkyl: saturated cyclic hydrocarbyl moieties, analogous to alkylgroups, having the indicated number of carbon atoms. Examples includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl andcylooctyl.

Alkenyl: Unsaturated acyclic hydrocarbyl moieties, with straight orbranched chains, containing one or more double (olefinic) bonds, andhaving the indicated number of carbon atoms. Examples include vinyl,allyl, isopropenyl, 2-butenyl, 1,3-butadienyl, and the various pentenyl,hexenyl and octenyl groups.

Alkyl, alkenyl cycloalkyl and other aliphatic groups may beunsubstituted or may be substituted. Typical substituents include halo,hydroxy, cyano, nitro, COOH and COOCH₃. Substituted moieties may havefrom one to as many substituents as are possible on the group inquestion. Preferably, substituted alkyl, alkenyl and cycloalkyl moietieshave from 1 to 4 substituents (of course, with a maximum number ofsubstituents possible for the group in question). In polysubstitutedcompounds the substituents may be the same or different, i.e. an alkylgroup may be substituted with two or three different halogens, or withhalo and hydroxyl groups.

Aralkyl: as generally used, refers to an alkyl group having an arylsubstituent. Aralkyl groups in compounds of the present invention andtheir compositions and uses have the general formula (CH₂)₁₋₄Ph₁₋₂ wherePh stands for phenyl. That is, they have from 1 to 4 methylene groups ina chain, substituted by one or two phenyl groups. An example of such anaralkyl group is 3,3-diphenylpropyl.

Halo includes fluoro, chloro, bromo and iodo substituents as indicated.Where a moiety or compound includes multiple halogens, they may be thesame or different; i.e. such a compound or moiety may contain two ormore different halogen atoms.

Fused carbocyclic ring moieties include fully or partly unsaturatedrings such as naphthyl, tetrahydronapthyl and phenyl substituted byalkylene groups having 2-4 carbon atoms. One example of thelast-mentioned type of fused ring is indanyl, i.e. a phenyl ringsubstituted with a propylene (—CH₂CH₂CH₂—) moiety.

Heterocyclic moieties include both saturated and unsaturated cyclicalmoieties having the indicated number of members, or atoms, including oneor more nitrogen, sulfur and/or oxygen atoms, as indicated. Theremaining atoms in the ring are carbon atoms. The moieties may containthe atoms in a single ring or in a fused ring. Examples of five-memberedheterocyclic rings include thienyl, furyl, tetrahydrofuryl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, thiazolidinyl, pyrrolyl,pyrrolidinyl, pyrrolinyl, imidazolyl, imidazolidinyl, pyrazolyl,triazolyl, and pyrazolidinyl. Examples of six-membered heterocyclicrings include pyridyl, pyrazinyl, pyrimidinyl, triazinyl, piperidyl,morpholinyl, pyranyl, tetrahydropyranyl, and piperazinyl.

Examples of heterocyclic moieties having more than six carbons includeindolyl, quinuclidyl, quinolyl, chromanyl, benzimidazolyl, benzoxazolyl,benzothienyl, benzofuranyl, and quinolinyl.

Heterocyclic moieties may be unsubstituted or may be substituted, forinstance, by from 1 to 3 groups independently selected from halo, C₁-C₄alkyl, hydroxy, or oxo, including combinations of such substituents.

Unsaturated moieties include in the case of carbocyclic and heterocyclicrings partially unsaturated moieties such as 1,2,3,4-tetrahydropyndinyland 2,3-dihydroindolyl, and fully unsaturated moieties such as pyridinyland indolyl.

Description of Particular Embodiments of the Invention

The general methods comprise the steps of contacting the receptor withan OX2R agonist of formula I, and detecting a resultant agonizing oractivation of the receptor. The contacting is generally effected byadministering to a person an effective amount of one or more compoundshaving the general formula I:

in which:

R₁, is

substituted or unsubstituted alkyl;

substituted or unsubstituted cycloalkyl;

substituted or unsubstituted alkenyl;

adamantyl;

substituted or unsubstituted phenyl;

a 5- or 6-membered optionally substituted saturated or unsaturatedheterocyclic group having from one to three heteroatoms selected fromnitrogen, oxygen and sulfur;

substituted or unsubstituted benzyl;

a saturated or unsaturated fused ring carbocyclic group having from 8 to10 ring atoms; or

(i) CH₂XR₅, where X is oxygen, sulfur, —NH— or —CH₂— and R₅ issubstituted or unsubstituted alkyl; substituted or unsubstitutedcycloalkyl; substituted or unsubstituted phenyl; substituted orunsubstituted benzyl; 2-carbamide-indolyl; or a 5 to 9-memberedoptionally substituted saturated or unsaturated heterocyclic grouphaving from one to three heteroatoms selected from nitrogen, oxygen andsulfur;

R₂ is

substituted or unsubstituted alkyl;

substituted or unsubstituted cycloalkyl;

substituted or unsubstituted alkenyl;

substituted or unsubstituted alkoxyalkyl;

a 5- or 6-membered optionally substituted saturated or unsaturatedheterocyclic group having from one to three heteroatoms selected fromnitrogen, oxygen and sulfur;

—(CH₂)_(n)R₃, where R₃ is (i) a 5- to 9-membered optionally substitutedsaturated or unsaturated heterocyclic group having from one to threeheteroatoms selected from nitrogen, oxygen and sulfur; (ii) —NR₆R₇ whereR₆ and R₇ are independently selected from hydrogen, methyl, ethyl andbenzyl; or (iii) COOR₈ where R₈ is alkyl; and n is 2 or 3;

substituted or unsubstituted phenyl;

substituted or unsubstituted benzyl;

a saturated or unsaturated fused ring carbocyclic group having from 8 to10 ring atoms; or

methylene-1-naphthyl; and

R₄ is

hydrogen;

(CH₂)_(m)COOR₁₅ where R₁₅ is alkyl or substituted alkyl; and m is 0, 1or 2;

CONR₁₆R₁₇, where R₁₆ and R₁₇ are independently (i) hydrogen; (ii) alkylor substituted alkyl; (iii) cycloalkyl; (iv) alkoxyalkyl; (v) a 5- to10-membered optionally substituted saturated or unsaturated heterocyclicgroup having from one to three heteroatoms selected from nitrogen,oxygen and sulfur; (vi) a saturated or unsaturated fused ringcarbocyclic group having from 8 to 10 ring atoms; (vii) substituted orunsubstituted phenyl; (viii) (CH₂)_(p)R 18 where R₁₈ is a 5- or6-membered optionally substituted saturated or unsaturated heterocyclicgroup having from one to three heteroatoms selected from nitrogen,oxygen and sulfur, and p is 1, 2 or 3; (ix) optionally substitutedbenzyl; or (x) an aralkyl group comprising a chain of from 1 to 4methylene groups substituted by one or two phenyl groups;

C₁-C₄ alkoxy;

optionally substituted phenoxy;

SO₂NR₁₉R₂₀ where R₁₉ and R₂₀ are independently hydrogen, optionallysubstituted alkyl or phenyl;

NR₂₁R₂₂;

COR₂₃ where R₂₃ is alkyl or is NR₂₁R₂₂;

(j) COOR₂₃ where R₂₃ is hydrogen, alkyl, or benzyl; or

SO₂R₂₅ where R₂₅ is alkyl or NR₂₁R₂₂; wherein R₂₁ and R₂₂ areindependently hydrogen, alkyl, optionally substituted phenyl oroptionally substituted benzyl;

or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In another embodiment the OX2R agonist is of the same general formula I(supra) in which:

R₁ is

C₁-C₆ alkyl, optionally substituted with one or more groups selectedfrom halo,

hydroxy, cyano, nitro, COOH and COOCH₃;

C₂-C₆ alkenyl, optionally substituted with one or more groups selectedfrom halo,

hydroxy, cyano, nitro, COOH and COOCH₃;

C₃-C₆ cycloalkyl, optionally substituted with one or more groupsselected from

halo, hydroxy, cyano, nitro, COOH and COOCH₃;

adamantyl;

optionally substituted phenyl in which the substituents are selectedfrom mono and dihalo, mono- and di-(C₁-C₄ alkoxy), mono- and di-(C₁-C₄alkylthio), C₁-C₂ alkyleneoxy; C₁-C₂ alkylenedioxy, mono- and di-(C₁-C₄alkyl), mono- and di-(trifluoromethyl), mono- and di-(C₁-C₄hydroxyalkyl), mono- and di-(C₁-C₄ alkoxyalkyl), mono- and di-hydroxy,mono- and di-cyano, mono- and di-(COCH₃), and mono- and di-NHR₂₆,wherein R₂₆ is COCH₃, SO₂CH₃, SO₂C₆H₅, COOR′ or CONR′R″; and wherein R′and R″ are independently hydrogen or C₁-C₄ alkyl;

a 5- or 6-membered saturated or unsaturated heterocyclic group havingfrom one to three heteroatoms selected from nitrogen, oxygen and sulfur,optionally substituted by

from 1 to 3 groups independently selected from halo, C₁-C₄ alkyl, C₁-C₄alkoxy, oxo and hydroxy;

optionally substituted benzyl in which the substituents are selectedfrom mono- and

dihalo, mono- and di-(C₁-C₄ alkoxy), mono- and di-(C₁-C₄ alkylthio),C₁-C₂ alkyleneoxy; C₁-C₂ alkylenedioxy, mono- and di-(C₁-C₄ alkyl),mono- and di-(trifluoromethyl), mono- and di-(C₁-C₄ hydroxyalkyl), mono-and di-(C₁-C₄ alkoxyalkyl), mono- and di-hydroxy, mono- and di-cyano,mono- and di-(COCH₃), and mono- and di-NHR₂₆;

naphthyl; or

CH₂XR₅, where X is oxygen, sulfur, —NH— or —CH₂— and R₅ is selected from(i) C₁-C₆ alkyl; (ii) C₃-C₆ cycloalkyl; (iii) optionally substitutedphenyl in which the substituents are selected from mono- and di-(C₁-C₄alkyl), mono- and dihalo, mono and di-(C₁-C₄ alkoxy), C₁-C₂ alkyleneoxy,C₁-C₂ alkylenedioxy, mono- and di-(trifluoromethyl), nitro, hydroxy,mono- and di-(C₁-C₄ hydroxyalkyl), mono- and di-(C₁-C₄ alkoxyalkyl),mono- and di-hydroxy, mono- and dicyano, mono- and di-(COCH₃) and mono-and di-NHR₂₆; (iv) benzyl; (v) 2-carbamide-indolyl; or (vi) a 5- to9-membered saturated or unsaturated heterocyclic group having from oneto three heteroatoms selected from nitrogen, oxygen and sulfur,optionally substituted by from 1 to 3 groups independently selected fromhalo, C₁-C₄ alkyl, C₁-C₄ alkoxy, oxo and hydroxy;

R₂ is

C₁-C₁₂ alkyl, optionally substituted with one or more groups selectedfrom halo, hydroxy, cyano, nitro, COOH and COOCH₃;

C₂-C₈ cycloalkyl, optionally substituted with one or more groupsselected from halo, hydroxy, cyano, nitro, COOH and COOCH₃;

C₂-C₁₂ alkenyl, optionally substituted with one or more groups selectedfrom halo, hydroxy, cyano, nitro, COOH and COOCH₃;

C₂-C₁₂ alkoxyalkyl;

a 5- or 6-membered saturated or unsaturated heterocyclic group havingfrom one to three heteroatoms selected from nitrogen, oxygen and sulfur,optionally substituted by from 1 to 3 groups independently selected fromhalo, C₁-C₄ alkyl, C₁-C₄alkoxy, oxo, and hydroxy;

—(CH₂)nR₃, where R₃ is (i) a 5- to 9-membered saturated or unsaturatedheterocyclic group having from one to three heteroatoms selected fromnitrogen, oxygen and sulfur, optionally substituted by from 1 to 3groups independently selected from halo, C₁-C₄ alkyl, C₁-C₄ alkoxy, oxo,and hydroxy; (ii) —NR₆R₇ where R₆ and R₇ are independently selected fromhydrogen, methyl, ethyl and benzyl; or (iii) COOR₈ where R₈ is C₁-C₄alkyl; and n is 2 or 3;

optionally substituted phenyl, where the substituents are independentlyselected from mono- and di-(C₁-C₄ alkyl); mono- and dihalo; mono-, di-,and tri-(C₁-C₄ alkoxy); mono- and dicyano; nitro; methylsulfonyl; mono-,di-, and tri-(trifluoromethyl); C₁-C₂ alkyleneoxy; C₁-C₂ alkylenedioxy;—(CH₂)_(q)COOR₉ where R₉ is C₁-C₄ alkyl; or —(CH₂)_(q)NR₁₀R₁₁ where R₁₀and R₁₁ are independently selected from hydrogen, C₁-C₄ alkyl, COR₁₂where R₁₂ is C₁-C₄ alkyl, SO₂R₁₃ where R₁₃ is C₁-C₄ alkyl, or COOR₁₄where R₁₄ is C₁-C₄ alkyl and CONR′R″; and q is an integer from 1 to 4;

optionally substituted benzyl, where the substituents are selected frommono-, di-, and tri-(C₁-C₄) alkyl; mono-, di-, and tri-halo; mono-, di-,and tri-(C₁-C₄ alkoxy); mono-, di-, and tri-(C₁-C₄ alkylthio); mono- anddi-cyano; nitro; methylsulfonyl; mono-, di-, and tri-(trifluoromethyl);mono-, di-, and tri-COOR′; mono-, di-, and triCONR′R″; mono-, di-, andtri-NR₂₇R₂₈ where R₂₇ and R₂₈ are independently selected from hydrogen,C₁-C₄ alkyl, acetyl, and methylsulfonyl; C₁-C₂ alkyleneoxy; C₁-C₂alkylenedioxy; and mono-, di-, and tri-(C₁-C₆ alkoxyalkyl);

a saturated or unsaturated fused ring carbocyclic group having from 8 to10 ring atoms; or

methylene-1- or 2-naphthyl;

and R₄ is

hydrogen;

(CH₂)_(m)COOR′ where m is 0, 1 or 2;

CONR₁₆R₁₇ where R₁₆ and R₁₇ are independently (i) hydrogen; (ii) C₁-C₅alkyl optionally substituted with one or more groups selected from halo,hydroxy, cyano, nitro, COOH and COOCH₃; (iii) C₃-C₆ cycloalkyl; (iv)C₂-C₈ alkoxyalkyl; (v) a 5- to 10-membered saturated or unsaturatedheterocyclic group having from one to three heteroatoms selected fromnitrogen, oxygen and sulfur, optionally substituted by from 1 to 3groups independently selected from halo, hydroxy, oxo, C₁-C₄ alkyl,C₁-C₄ alkoxy, COCH₃, COOR′, and NR₂₉R₃₀ where R₂₉ and R₃₀ areindependently selected from hydrogen, C₁-C₄ alkyl, acetyl, ormethylsulfonyl; (vi) a saturated or unsaturated fused ring carbocyclicgroup having from 8 to 10 ring atoms; (vii) (CH₂)_(p)R₁₈ where R₁₈ is a5- or 6-membered saturated or unsaturated heterocyclic group having from1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur, optionallysubstituted by from 1 to 3 groups independently selected from halo,hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy and oxo, and p is 1, 2 or 3; (viii)phenyl optionally substituted by one or more groups independentlyselected from mono-, di-, and tri-halo, mono-, di-, and trihydroxy,mono-, di-, and tri-(C₁-C₄ alkyl), C₁-C₂ alkyleneoxy, C₁-C₂alkylenedioxy, COOR′, and NR₂₉R₃₀ where R₂₉ and R₃₀ are independentlyselected from hydrogen, C₁-C₄ alkyl, acetyl, and methylsulfonyl; (ix)optionally substituted benzyl where the substituents are selected frommono-, di-, and tri-(C₁-C₄ alkyl); mono-, di-, and trihalo; mono-, di-,and tri-(C₁-C₄ alkoxy); nitro; methylsulfonyl; mono-, di-, andtri-(trifluoromethyl); NR′R″; C₁-C₂ alkyleneoxy; C₁-C₂ alkylenedioxy;(x) an aralkyl group comprising a chain of from 1 to 4 methylene groupssubstituted by one or two phenyl groups;

C₁-C₄ alkoxy;

optionally substituted phenoxy, where the substituents are independentlyselected from mono- and di-(C₁-C₄) alkyl; mono- and dihalo; mono-, di-,and tri-(C₁-C₄ alkoxy); cyano; nitro; methylsulfonyl; mono-, di-, andtri-trifluoromethyl; C₁-C₄ alkyleneoxy; C₁-C₂ alkylenedioxy;—(CH₂)_(r)COOR₉ where R₉ is C₁-C₄ alkyl; or (CH₂)_(r)NR₃₀R₃₁, where R₃₀and R₃₁ are independently hydrogen, C₁-C₄ alkyl, COR₃₂ where R₃₂ isC₁-C₄ alkyl, SO₂R₃₃ where R₃₃ is C₁-C₄ alkyl, or COOR₃₄ where R₃₄ isC₁-C₄ alkyl;

SO₂NR₃₅R₃₆ where R₃₅ and R₃₆ are independently hydrogen, C₁-C₄ alkyl orphenyl; or

NR₃₇R₃₈ where R₃₇ and R₃₈ are independently hydrogen; C₁-C₄ alkyl;phenyl; COR₃₉ where R₃₉ is C₁-C₄ alkyl; or SO₂R₄₀ where R₄₀ is hydrogenor C₁-C₄ alkyl;

and pharmaceutically acceptable salts thereof, and prodrugs thereof.

More particular embodiments include those:

in which R₁ is phenyl, substituted phenyl (compounds in which R₁ isunsubstituted phenyl and mono- or dihalophenyl being especiallypreferred), heterocyclic groups (thienyl being especially preferred), orCH₂XR₅, (especially preferred are those compounds where X is oxygen andR₅ is substituted or unsubstituted phenyl or is benzyl);

in which R₂ is benzyl, substituted benzyl, C₂-C₄ alkyl, or —(CH₂)₂R₃,where R₃ is a 5- to 6-membered saturated or unsaturated heterocyclicgroup having from one to three heteroatoms selected from nitrogen,oxygen and sulfur (2-N-piperidoethyl being most preferred of these);

and in which R₄ is hydrogen;

or in which R₁, R₂ and R₄ are combinations of these preferred subgroups.

Table 1 below includes representative compounds of this series. Asshown, the compounds in Table 1 are in the form of salts, particularlythe hydrochloride and hydrobromide salts. However, this was done forconvenience, and the invention is not limited to the use of these orother salts, but encompasses the compounds per se as well as theirpharmaceutically acceptable salts.

TABLE 1

Compound no./salt R1 R2 R4  1/hydrochloride —CH₂O(4-ClC₆H₄) benzyl H(4-chlorophenoxy-methyl)  2/hydrochloride 3,4-dichlorophenyl benzyl H 3/hydrochloride —CH₂OC₆H₅(phenoxymethyl) benzyl H  4/hydrochloridephenoxymethyl 4-methylbenzyl H  5/hydrochloride phenyl benzyl H 6/hydrochloride 2-thiophenyl benzyl H  7/hydrochloride 4-chiorophenylbenzyl H  8/hydrochloride phenoxymethyl n-butyl H  9/hydrochloride3,4-dichlorophenyl ethyl H 10/hydrochloride 4-chlorophenoxy-n-butyln-butyl H 11/hydrochloride 3,4-dichlorophenyl 4-methylbenzyl H12/hydrochloride phenoxymethyl 4-t-butylbenzyl H 13/hydrochloride3,4-dichlorophenyl methyl H 14/hydrochloride phenoxymethyl4-chlorobenzyl H 15/hydrochloride 4-chlorophenoxy-4- 4-chlorobenzyl Hchlorobenzyl 16/hydrochloride 2-thienyl 2-(1-morpholino)- H ethyl17/hydrochloride 2-thienyl ethyl H 18/hydrochloride phenyl n-butyl H19/hydrochloride phenoxymethyl ethyl H 20/hydrochloride phenoxymethyln-propyl H 21/hydrochloride p-chlorophenoxy-methyl ethyl H22/hydrochloride phenoxymethyl (1-piperido)ethyl H 23/hydrochloride1-adamantyl methyl H 24/hydrochloride methyl benzyl H 25/hydrochloride2-furyl 2-(1-morpholino)- H ethyl 26/hydrochloride t-butyl benzyl H27/hydrochloride 4-methoxyphenyl methyl H 28/hydrochloride4-methylphenyl 2-(N,N-diethyl- H amino)ethyl 29/hydrochloride4-chlorophenoxy-methyl n-propyl H 30/hydrochloride phenyl2-(1-piperido)ethyl H 31/hydrochloride 4-dimethoxyphenyl 2-(N,N-diethyl-H amino)ethyl 32/hydrochloride 1-naphthyl 2-(1-piperido)ethyl H33/hydrochloride phenoxymethyl 2-(1- H morpholino)ethyl 34/hydrochloride2-thienyl Methyl H 35/hydrochloride 4-chlorophenoxy-methyl 2-(1- Hmorpholino)ethyl 36/hydrochloride t-butyl allyl H 37/hydrochloride4-ethoxyphenyl 2-(1-piperido)ethyl H 38/hydrochloride 2-thienyl2-(1-piperido)ethyl H 39/hydrochloride 4-bromophenyl allyl H40/hydrochloride 4-chlorophenoxy-methyl 2-(1-piperido)ethyl H41/hydrochloride phenoxymethyl allyl H 42/hydrochloride4-chlorophenoxy-n-propyl 2-(N,N-diethyl- H amino)ethyl 43/hydrochloridephenyl n-propyl H 44/hydrochloride phenoxymethyl methyl H45/hydrochloride 4-methoxyphenyl 2-(1-piperido)ethyl H 46/hydrochloride4-chlorophenoxy-methyl allyl H 47/hydrochloride 4-methoxyphenyl benzyl H48/hydrochloride 4-chlorophenyl n-propyl H 49/hydrochloride4-ethoxyphenyl allyl H 50/hydrochloride 4-methylphenyl allyl H51/hydrochloride 3,4-dichlorophenyl 2-(N,N-diethyl- H amino)ethyl52/hydrochloride 4-chlorophenoxy-methyl 4-methylbenzyl H53/hydrochloride 4-chlorophenyl n-butyl H 54/hydrochloride4-chlorophenyl methyl H 55/hydrochloride phenoxymethyl 2-(N,N-diethyl- Hamino)ethyl 56/hydrochloride 4-chlorophenoxy-methyl 2-(N,N-diethyl- Hmethyl-aminoethyl) 57/hydrochloride 4-chlorophenoxy-methyl methyl H58/hydrochloride 1-naphthyl methyl H 59/hydrochloride t-butyl4-chlorobenzyl H 60/hydrochloride 4-methylphenyl n-propyl H61/hydrochloride methyl n-propyl H 62/hydrochloride 4-bromophenyln-butyl H 63/hydrochloride 4-bromophenyl benzyl H 64/hydrochloridep-chlorophenyl allyl H 65/hydrochloride phenyl methyl H 66/hydrochloridemethyl methyl H 67/hydrochloride 2-furyl benzyl H 68/hydrochloride2-furyl 4-chlorobenzyl H 69/hydrochloride t-butyl 2-(1-piperido)ethyl H70/hydrochloride phenoxymethyl 4-fluorobenzyl H 71/hydrochloride—CH₂OCH₂C₆H₅ benzyl H (benzyloxymethyl) 72/hydrochloride phenoxymethyln-octyl H 73/hydrochloride phenoxymethyl Methylene-1- H naphthyl74/hydrochloride phenoxymethyl n-undecyl H 75/hydrochloridephenoxymethyl benzyl —CO₂C₂H₅ 76/hydrochloride phenoxymethyl2-(N,N-dibenzyl- H amino)ethyl 77/hydrochloride benzyl benzyl H78/hydrochloride phenoxymethyl 4-methoxybenzyl H

Process: In general, the subject compounds may be prepared by a stepwisealkylation of 2-aminobenzimidazole or a ring-substituted2-aminobenzimidazole where R₄ is other than hydrogen.

In the first alkylation, the sodium salt of 2-aminobenzimidaZole reactssmoothly with alkyl and benzyl chlorides (Joseph, J Med Chem 28: 601(1963); Ogura et al, J Med Chem 15: 923-926 (1972)). The secondalkylation proceeds under conditions of high concentration in refluxingtoluene, reacting exclusively at the 3-position of the imidazole moiety(Rehse, et al., Arch Pharm (Weinheirn) 328: 77-80, 1995). Theprecipitation of the product as the hydrobromide salt prohibitsadditional alkylation and simplifies the purification.

Alternatively, as described below with respect to the preparation of acombinatorial library, the compounds can be prepared by a process inwhich a resin-bound 4-fluoro-3-nitroarene is reacted with an aminehaving the formula R2-NH2, reduced with tin (II) chloride (Bellamy, etal., Tetrahedron Lett 25: 839-842, 1984), cyclized with cyanogen bromide(U.S. Pat. No. 4,002,623), and reacted with a mono-substituted epoxide:

If R₄ is a group having the formula CONR₁₆R₁₇ that group is firstintroduced by amide coupling with the fluoronitrobenzoic acid.

The subject compounds are incorporated into pharmaceutical compositionsor formulations. The compositions will contain pharmaceuticallyacceptable diluents and/or carriers, i.e. diluents or carriers that arephysiologically compatible and substantially free from pathogenicimpurities. Suitable excipients or carriers and methods for preparingadministrable compositions are known or apparent to those skilled in theart and are described in more detail in such publications as Remington'sPharmaceutical Science, Mack Publishing Co, NJ (1991). The compositionsmay also be in the form of controlled release or sustained releasecompositions as known in the art. For many applications the subjectcompounds are administered for morning/daytime dosing, with off periodat night.

The subject compounds may be used per se, or in the form of theirpharmaceutically acceptable salts, such as hydrochlorides,hydrobromides, acetates, sulfates, citrates, carbonates,trifluoroacetates and the like. When compounds contain relatively acidicfunctionalities, salts can be obtained by addition of the desired base,either neat or in a suitable inert solvent. Examples of pharmaceuticallyacceptable base addition salts include sodium, potassium, calcium,ammonium, organic amino, or magnesium salts, or the like. When compoundscontain relatively basic functionalities, salts can be obtained byaddition of the desired acid, either neat or in a suitable inertsolvent. Examples of pharmaceutically acceptable acid addition saltsinclude those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galacturonic acids and thelike (see, for example, Berge et al, “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19).

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid, and isolating the parent compound in theconventional manner The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of this invention.

In addition to salt forms, this invention provides compounds which arein a prodrug form. Prodrugs of the compounds described herein are thosecompounds that readily undergo chemical changes under physiologicalconditions to provide the compounds of the present invention.Additionally, prodrugs can be converted to the compounds of the presentinvention by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to the compounds of thepresent invention when placed in a transdermal patch reservoir with asuitable enzyme or chemical reagent. Prodrugs are often useful because,in some situations, they may be easier to administer than the parentdrug. They may, for instance, be more bioavailable by oraladministration than the parent drug. The prodrug may also have improvedsolubility in pharmacological compositions over the parent drug. A widevariety of prodrug derivatives are known in the art, such as those thatrely on hydrolytic cleavage or oxidative activation of the prodrug. Anexample, without limitation, of a prodrug would be a compound of thepresent invention which is administered as an ester (the “prodrug”), butthen is metabolically hydrolyzed to the carboxylic acid, the activeentity.

Certain compounds of the invention can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of theinvention.

Some of the subject compounds possess asymmetric carbon atoms (opticalcenters) or double bonds; the racemates, diastereomers, geometricisomers and individual isomers are all intended to be encompassed withinthe scope of the present invention.

The compounds of the invention may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe invention, whether radioactive or not, are intended to beencompassed within the scope of the invention.

The compounds are generally administered in a “therapeutically effectiveamount”, i.e. the amount of the subject compound that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician. The term “therapeutically effective amount” includes thatamount of a compound that, when administered, is sufficient to preventdevelopment of, or alleviate to some extent, one or more of the symptomsof the condition or disorder being treated. The therapeuticallyeffective amount will vary depending on the compound, the disease andits severity and the age, weight, etc., of the mammal to be treated.

The contacting is generally effected by administering to the subject aneffective amount of one or more compounds having the general formula I(supra), including the various embodiments described above. Generallyadministration is adjusted to achieve a therapeutic dosage of about 0.1to 50, preferably 0.5 to 10, more preferably 1 to 10 mg/kg, thoughoptimal dosages are compound specific, and generally empiricallydetermined for each compound.

The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient. Typical unit dosage forms includeprefilled, premeasured ampules or syringes of the liquid compositions orpills, tablets, capsules, lozenges or the like in the case of solidcompositions. In such compositions, the mimetic is usually a minorcomponent (from about 0.1 to about 50% by weight or preferably fromabout 1 to about 40% by weight) with the remainder being variousvehicles or carriers and processing aids helpful for forming the desireddosing form. Unit dosage formulations are preferably about of 5, 10, 25,50, 100, 250, 500, or 1,000 mg per unit. In a particular embodiment,unit dosage forms are packaged in a multipack adapted for sequentialuse, such as blisterpack comprising sheets of at least 6, 9 or 12 unitdosage forms.

The subject compositions may also be coformulated and/or coadministeredwith a different compound which also promotes wakefulness, anti-obesity,and/or recovery from general anesthesia or jet lag. Example ofco-formulatable drugs include diet pills such as Orlistat (Xenical);narcolepsy drugs such as methylphenidate, racemic amphetamine,dextroamphetamine, and methamphetamine, or modafinil; stimulants such ascaffeine; etc.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those skilled in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example 1 Synthesis of1-benzyl-2-imino-3-(2-hydroxy-3-phenoxpropyl)benzimidazole hydrobromide(1) (Compound #3)

To a solution of 2-aminobenzimidazole (2) (Aldrich, 6 g., 45 mmol) inEtOH (10 mL) is added EtONa (21% in EtOH, 16.83 mL, 45 mmol) and benzylchloride (5.70 mL, 50 mmol) and the brown solution is refluxed for 3days under N₂ gas.

After cooling to room temperature, the reaction mixture is filteredthrough celite and concentrated in vacuo. The resulting brown solid isfiltered hot in 300 mL acetone and crystallized from approximately 100mL of acetone, and gives 3 as brown crystals (3.91 g., 39%).Recrystallization of the mother liquor affords additional 3 as browncrystals (0.70 g., 7%). ¹H-NMR (DMSO-d₆) δ 5.27 (s, 2 H), 6.61 (s, 2 H),6.82 (t, J=8.8 Hz, I H), 6.93 (t, J=6.4 Hz, 1 H), 7.05 (d, J=8.0 Hz, 1H), 7.15 (d, 7.2 Hz, 1 H), 7.20 (d, 6.8 Hz, 2 H), 7.24 (d, 7.6 Hz, I H),7.31 (t, 7.6 Hz, 2 H); ¹³C-NMR (DMSO-d₆) 45.11, 108.30, 115.19, 118.49,120.88, 127.39, 127.66, 128.91, 134.60, 137.64, 143.34, 155.46.

To a room temperature water cooled solution of allyl phenyl ether (4)(Aldrich, 1.00 g., 7.45 mmol) in 5 mL DMSO: H₂O (4:1) is added NBS (1.45g., 8.15 mmol) as a solid. After 5-10 minutes, the reaction is added toa separation funnel with 100 mL Et₂O and washed three times with 100 mLwater, then 100 mL brine solution. The ether layer is dried over MgSO₄and concentrated in vacuo to yield a pale yellow oil (1.59 g., 92%),which is used as is without further purification.

To a solution of 1-bromo-3-phenoxy-propan-2-ol (5) (248 mg, 1.07 mmol)in toluene (2 mL) is added 2-amino-1-benzylbenzimidazole (3) (200 mg,0.90 mmol) and the mixture is heated at reflux overnight under N₂ gas.The reaction mixture is cooled to room temperature and filtered.Crystallization of the collected solid from isopropanol gives 1 as whitecrystals (85 mg, 23%). Recrystallization of the mother liquor affordsadditional 1 as white crystals (70 mg., 19%). ¹H-NMR (DMSO-d₆) δ 4.12(s, 2 H), 4.33 (s, 1 H), 4.43 (s, 2 H), 5.57 (s, 2H), 6.96 (m, 3 H),7.21-7.38 (m, 10 H), 7.46 (d, J=8.0 Hz, IH), 7.62 (d, J=7.6 Hz, 1H),9.18 (s, 2 H); ¹³C-NMR (DMSO-d₆) δ 45.58, 46.10, 66.54, 69.43, 110.53,111.11, 120.71, 123.39, 123.47, 127.11, 127.91, 128.72, 129.39, 129.45,130.39, 134.56, 150.27, 157.67, 158.33.

Example 2

Preparation of Library of Compounds Using a Combinatorial Method.

A library of compounds in which R₄ was various groups having the formulaCONHR is prepared by the process described above using4-fluoro-3-nitrobenzoic acid, as follows:

Aldehyde resin is mixed with a primary amine (R₁₇—NH₂) indichloroethane(DCE), triethylorthoformate (TEOF), and DMF (containing 1% acetic acid)in a 1:1:1 ratio. After shaken overnight, sodium triacetoxyborohydride(20 eq.) dissolved in DMF is added (Abdel-Magid, et al., TetrahedronLett, 3 1: 5595-5598, 1990). After the mixture is shaken at roomtemperature overnight, the resin is filtered and washed with DMF (3×5mL), MeOH (3×5 mL), DMF (3×5 mL), MeOH (3×5 mL), and CH₂Cl₂ (3×5 mL).The resin was washed twice with 5 mL DMF containing 1% Hunig's base. Tothe filtered resin is added a mixture of 4-fluoro-3-nitrobenzoic acid(FNBA, 10 eq.) and diisopropylcarbodiimide (DIC, 5 eq.) in 2:1 DMF:DCM.After shaking at room temperature overnight, the resin is filtered andwashed with DMF (3×5 mL) and CH₂Cl₂ (3×5 mL).

The resin is shaken with a primary amine (R₂—NH₂) in DMF for 8 hrs,filtered, and washed with DMF (6×5 mL), MeOH (3×5 mL), and CH₂Cl₂ (3×5mL). The aryl nitro group is reduced by the addition of tin (II)chloride dihydrate (20 eq, >2 M) and N-methyl morpholine (NMM, 20 eq.)in N-methylpyrrolidinone (NMP). After shaken at room temperatureovernight, the resin is filtered and washed with NMP (3×5 mL), MeOH (3×5mL), and CH₂Cl₂ (3×5 mL). The resulting resin is shaken at roomtemperature with cyanogen bromide (5 eq.) overnight, filtered, andwashed with CH₂Cl₂ (3×5 mL), MeOH (3×5 mL), and CH₂Cl₂ (3×5 mL). Toproduce a free amine, the resin is shaken for 30 min. in CH₂Cl₂ with theaddition of sodium methoxide in methanol, filtered, and washed withCH₂Cl₂ (4×5 mL).

In the final diversification step, the resin is heated at 500 C. in DMFwith a mono-substituted epoxide [R1CH(—CH₂O—)]. After shaking for 2 to 4days the resin is filtered and washed with DMF (5×5 mL), MeOH (3×5 mL),and CH₂Cl₂ (3×5 mL). The resin-bound benzimidazole is cleaved from thesolid-support by treatment with TFA: CH₂Cl₂ (2:3) for 1 hour at roomtemperature. The library contains a total of 10,560 compounds, preparedusing 32 species of group R₁₇, 33 of group R₂ and 10 of group R₁. Thelibrary of compounds is depicted in Table 2:

TABLE 2

R # Structure Group Common Name 1 —CH₃ 17 methyl 2 —CH₂—CH₃ 17 ethyl 3—CH₂CH₂—CH₃ 17 propyl 4

17 3-pyrazolyl 5

17 cyclopentyl 6 -Ph 17 phenyl 7

17 2-pyrimidinyl 8

17 furfuryl 9

17 tetrahydrofurfuryl 10

17 3-keto-4,5-dihydroisoxazolyl 11 —CH₂CH₂OCH₂CH₂CH₃ 17 propoxyethyl 12-Bn 17 benzyl 13

17 picolyl 14

17 2-thienylmethyl 15 —CH₂CH₂N(CH₂)₄ 17 N-ethyl-pyrrolidine 16—CH₂CH₂COOCH₂CH₃ 17 propoxylate ethyl ester 17 -Bn-4-Me 174-methylbenzyl 18

17 2-(1-methyl-pyrrolidinyl)ethyl 19

17 2-(1-morpholino)ethyl 20 -Bn-3-OMe 17 3-methoxybenzyl 21 -Bn-3-Cl 173-chlorobenzyl 22

17 3-(1-pyrrolidinyl-2-one)propyl 23 -Bn-4-NMe₂- 174-(dimethylamino)benzyl 24

17 piperonyl 25 -Bn-4-NO₂— 17 4-nitrobenzyl 26

17 3-(4-methyl-1-piperazinyl)propyl 27

17 2-methyl-4-quinolinyl 28 -Bn-3-CF₃ 17 3-trifluoromethylbenzyl 29-Bn-2,6-Cl 17 2,6-dichlorobenzyl 30 -Bn-4-SO₂Me 174-(methylsulfonyl)-benzyl 31 -Bn-3,4,5-OMe 17 3,4,5-trimethoxy-benzyl 32—CH₂CH₂CHPh₂ 17 3,3-diphenylpropyl 1 —CH(CH₃)₂ 2 isopropyl 2 —CH₂CH₂OH 22-hydroxyethyl 3

2 3-pyraxolyl 4 —CH₂CH₂CH(CH₃)₂ 2 isopentyl 5 -Ph 2 phenyl 6

2 3-pyridyl 7

2 2-pyrimidinyl 8

2 furfuryl 9

2 cyclohexyl 10

2 N-piperidinyl 11

2 tetrahydrofurfuryl 12 —CH₂CH₂OCH₂CH₂CH₃ 2 propoxyethyl 13 -Bn 2 benzyl14

2 2-picolyl 15

2 2-thienylmethyl 16 —CH₂CH₂N(CH₂)₄ 2 2-(1-pyrrolidinyl)ethyl 17—CH₂CH₂COOCH₂CH₃ 2 propoxylate ethyl ester 18 -Bn-4-Me 2 4-methylbenzyl19

2 3-quinuclidinyl 20

2 2-(1-piperidino)ethyl 21

2 2-(1-morpholino)ethyl 22

2 1-idanyl 23 -Bn-3-OMe 2 3-methoxybenzyl 24 -Bn-3-Cl 2 3-chlorobenzyl25

2 3-(1-morpholino)propyl 26 -Bn-4-NMe₂ 2 4-(dimethylamino)benzyl 27

2 piperonyl 28 —BN-4-NO₂ 2 4-nitrobenzyl 29

2 3-(4-methyl-1-piperazinyl)propyl 30 -Bn-3-CF₃ 23-trifluoromethylbenzyl 31 -Bn-2,4-Cl 2 2,4-dichlorobenzyl 32-Bn-4-SO₂Me 2 4-(methylsulfonyl)benzyl 33 -Bn-3,4,5-OMe 23,4,5-trimethoxybenzyl 1 —CH₂OPh 1 phenoxymethyl 2 —CH₂OCH(CH₃)₂ 1isopropoxymethyl 3 —CH₂OPh-4-OMe 1 4-methoxy-phenoxymethyl 4—CH₂OPh-4-C(CH₃)₂ 1 4-t-butyl-phenoxymethyl 5

1 2-furfuryloxymethyl 6 —CH₂OPh-2-Me 1 2-methyphenoxymethyl 7—CH₂OPh-4-Cl 1 4-chlorophenoxymethyl 8 -Ph 1 phenyl 9

1 2-carbamyl-5-inolyl-oxymethyl 10 —CH₂OPh-4-NO₂ 1 4-nitrophenoxymethyl

Example 3 Cell-based Transcription Assay for OX2R Agonists

DNA Construction: Open reading frames of OX1R, OX2R and GRP (control)receptor were inserted into a bicistronic retrovirus expression vector,pMXs-IRES-puror (pMXs-IP), which carried a FLAG™ epitope cDNA sequenceat the beginning of the ORF sequences encoding their human polypeptides.

Transfection and retrovirus production: Transfection was carried outusing FuGENE 6 (Roche Diagnostics Corp.) according to the manufacturer'srecommendations. Retroviruse fluid was harvested after 48 hours posttransfection.

Cell lines: To generate receptor expressing cells, CHO cells harboringecotropic retroviral receptor were infected with a retroviralsupernatant. After 24 hours post infection, the retrovirus supernatantwas removed and infected cells were selected with 10 μg/ml of puromycin(Sigma-Aldrich, St. Louis, Mo.).

To generate stable indicator cell lines, constitutively receptorexpressing CHO cell lines were transfected with NFAT-responsiveluciferase reporter plasmid (pNFAT-TA-Luc) together with pSV2neo whichcarried antibiotic resistance gene, by using FuGENE6 (Roche Diagnostics,Roskilde, Denmark). After 24 hours, the transfected cells were selectedwith 1 mg/ml G418 (invitrogen). G418 resistance cells were seeded andisolated single clones.

Luciferase reporter assay: The indicator cells were seeded at 5,000/wellin a 384-well plate and incubated at 37° C. for overnight. 2.5 μl oflibrary compound was added in each wells and incubated at 37° C. for 7-8hours. After removed media, cells were lysed and determined theluciferase activity with Bright-Glo™ (Promega, Madison, Wis.).

Intracellular Calcium Transient Assay: The CHO cell lines expressingappropriate G-protein coupled receptors were incubated with 2 μM Fura-2AM (Invitrogen, Carlsbad, Calif.) in a loading buffer (Hank's balancedsalt solution containing 25 mM HEPES, 2.5 mM probenecid and 0.1% bovineserum albumin) at room temparature for 1 hour. The cells were harvestedby centrifugation and washed the cell pellet with the loading bufferthree times, then suspended in the loading buffer at 2×10⁶ cells/ml.

0.5 ml aliquot of the cell suspension was stimulated by addition of 5 μlof compound dissolved in DMSO. Changes of [Ca²⁺]i transients in cellswere measured at RT by using CAF-110 Intracellular Ion Analyser (JASCO,Tokyo) with the excitation wave lengths of 340 nm and 380 nm andemission wavelength of 500 nm.

Example 4

Identification and Characterization of Cyclic Guanidinyl Compound No.130699

Compound 130699 (also referred to as #6037874 and Yan 7874) inducesrobust Ca2+response in OX2R-expressing CHO cells, a weaker response inOX1R-expressing cells, and no response in cells expressing an unrelatedGq-coupled GPCR (GRP receptor). This compound also induces dramaticactivation in transcriptional reporter assays in HEK293T cellstransiently co-transfected with the NFAT-luciferase reporter plasmid anda receptor cDNA plasmid. We also confirmed reporter activation in adistinct CRE reporter system:

OX2R_NFTA OC2R_NFTA No Assay (1) (2) OX2R_CRE Receptor_CRE Fold 10.9435.566 5.1935 0.797 Induc- tion

Metabolism: 2 μM Yan7874 was incubated in 1 ml 0.1M Tris/0.5% NaHCO₃/3mm MgCl₂ solution with 1 mg mouse CD-1 S9 protein (In VitroTechnologies, Baltimore, Md.) at 37° C. Cofactors necessary for phase Iand II metabolism were added as recommended by In Vitro Technologies(phase I: 1.7 mg/ml NADP, 7.8 mg/ml glucose-6-phophate, 6 U/mlglucose-6-phosphate dehydrogenase; phase II additionally add 1.9 mg/mluridine 5′-diphospho-α-D-glucuronic acid and 100 μg/ml3′-phosphoadenosine-5′-phophosulphate). Protein was precipitated by theaddition of 1 volume of methanol. Samples in 50:50 buffer:methanol+0.1%added formic acid were analyzed as follows. An analytical method wasdeveloped to detect Yan7874 using LC/MS/MS. Yan7874 was detected in SIMmode using the 426.0 to 105.0 transition (426.0 protonated parention/105.0 daughter ion) and had a retention time of 6.35 minutes whenrun on a 12 minute water/methanol (+0.1% formic acid) gradient (0 to100% methanol) using a Phenomenex synergi fusion RP column (5 micronpacking, 75×2 mm size). The fraction of parent compound remaining wasplotted over time and a curve fit to the data. The time at which 50% ofcompound remains is designated the in vitro half-life. See FIG. 1showing that compound 130699 (referred to as Yan7874 here) is relativelystable in vitro using hepatocyte S9 fractions.

Pharmacokinetics: Methods: Chembridge Compound 6037874 (“Yan7874”) wasprovided as a powdered stock and dissolved in DMSO at 50 mg/ml. Thecompound was formulated as 5% DMSO/5% Cremaphor EL/90% 5% dextrose, pH7.2 such that compound could be administered to mice at 10-20 mg/kg in avolume of 0.2 ml by IV bolus.

Twenty one C57BL/6 mice (NCI, male, 6 weeks of age) were weighed andinjected with 10 mg/kg compound formulated as described above IV in 0.2ml. A dose of 20 mg/kg was tried initially, but the first two miceinjected at this dose showed a relatively severe reaction marked by amild seizure and reduced activity for several hours. The dose waslowered to 10 mg/kg and no acute reactions were noted, so the remainderof the experiment was conducted at this dose. The mice were sacrificedin groups of three by inhalation overdose of CO2 and blood and brainobtained. Plasma was subsequently isolated from the plasma bycentrifugation in a refrigerated microcentrifuge at 10,000 rpm for 10′.The brain was washed extensively with PBS, blotted dry, weighed, andsnap frozen in liquid nitrogen.

A second experiment was conducted using 24 C57BL/6 mice (NCl, male, 6weeks of age). These mice were weighed and injected with 20 mg/kg ofcompound formulated as described above IP in 0.2 ml. A dose of 30 mg/kgwas tried initially, but one of the two mice injected at this doseappeared to show abdominal guarding and decreased activity for up to onehour. The dose was lowered to 20 mg/kg and no acute reactions werenoted, so the remainder of the experiment was conducted at this dose.The mice were sacrificed as above in groups of three by inhalationoverdose of CO2 and blood and brain obtained and processed as above.

An analytical method was developed to detect Yan7874 using LC/MS/MS.Yan7874 was detected in SIM mode using the 426.0 to 105.0 transition(426.0 protonated parent ion/105.0 daughter ion) and had a retentiontime of 6.35 minutes when run on a 12 minute water/methanol (+0.1%formic acid) gradient (0 to 100% methanol) using a Phenomenex synergifusion RP column (5 micron packing, 75×2 mm size).

A standard curve was developed in plasma in the following manner. 100 μlof plasma is spiked with varying concentrations of Yan7874. The plasmais allowed to sit 10′ at room temperature after vortexing. 200 μl ofacetonitrile is added to precipitate plasma proteins and release bounddrug. After vortexing, the sample is incubated 10′ at room temperature.700 μl of PBS is added and the sample is centrifuged at 14,000 rpm in astandard microcentrifuge for 5′. 900 μl of supernatant is added to 1 mlof PBS. The entire sample is passed over a Waters OASIS HLB solid phasedextraction column already primed by addition of 2 ml methanol and 2 mlH₂O. The column is washed once with 2 ml of 5% methanol/2% NH4OH in H2Oand once with 2 ml of 5% methanol/2% acetic acid in H₂O and compound iseluted by addition of 2 ml of methanol containing 2% NH₄OH. 500 μl ofthis is added to 500 μl of H20 containing 0.2% formic acid (final formicacid 0.1%). Samples are prepared in the same manner without addition ofspiked compound. Brain tissue is prepared by homogenizing in 3× volumeof PBS (weight in grams×3=volume in ml to add). 100 μl of blank brainlysate is used for preparation of standard curve samples while 100 μl ofsample brain lysate is used for preparation of samples. To calculatebrain levels, the concentration in ng/ml calculated from the standardcurve is multiplied by the total volume of lysate (a value in mlestimated from the 3× volume of PBS added+1× volume to account for braintissue itself) and then divided by the weight of the brain in grams. Avalue of 3× above the signal obtained from blank plasma or brain lysateis designated the limit of detection (LOD). The LOD for plasma is 2640and for brain 459. The limit of quantitation (LOQ) is the lowestconcentration at which back calculation yields a concentration within20% of theoretical. The LOQ for both plasma and brain is 5 ng/ml. Ingeneral back calculation of points on both curves yielded values within20% of theoretical over 4 orders of magnitude (10,000 to 5 ng/ml) forboth sample types.

Results: The compound shows good brain penetration, as predicted, withlevels of 5 ng/g persisting at 24 hours post-injection and peak levelsof ˜2.5 μg/g brain tissue achieved 30 minutes after dosing (see FIG. 2).Indeed, its peak brain concentrations (which occur ˜30 min later) are ˜5fold higher than the peak levels in plasma. The pharmacokinetic datawere modeled for the plasma data using the noncompartmental model ofWinNonlin (sparse sampling option), with the following parametersestimated:

IV Pharmacokinetics (10 mg/kg) IP Pharmacokinetics (20 mg/kg) Plasma:Plasma: AUC*: 91803 min * ng/ml AUC: 143,707 min * ng/ml Clearance: 2.1ml/min Clearance: 2.7 ml/min Terminal half life: 91.3 min Terminal halflife: 165 min Cmax: 512 ng/ml Cmax: 1076 ng/ml Brain: Brain: AUC 485,627min * ng/g AUC 133,194 min * ng/g Clearance: 0.4 g/min Clearance: 2.9g/min Terminal half life: 187.5 min Terminal half life: 203.5 min Cmax:2481 ng/g Cmax: 337 ng/g Blood Brain Penetration Ratio: Blood BrainPenetration Ratio: Cmax brain/Cmax plasma: 4.8 Cmax brain/Cmax plasma:0.3 AUC brain/AUC plasma: 5.2 AUC brain/AUC plasma: 0.9 *Area under theconcentration-time curve

Example 5

SAR Studies Around the Cyclic Guanidinium Scaffold of Compound 130699

The half-life of compound 3a (FIG. 3) for in vitro Phase I and combinedPhase I+II metabolism in S9 fractions was 68 and 56 min respectively.Most importantly, compound 3a shows good brain penetration, with levelsof 5 ng/g persisting at 24 hours post-injection and peak levels of ˜2.5μg/g brain tissue achieved 30 minutes after dosing (21 mice, dosing: 10mg/Kg iv). Plasma pharmacokinetic data were excellent: Area under theconcentration-time curve (AUC)=91803 min*ng/ml; Clearance=2.1 ml/min;Terminal half-life=91.3 min

We initiated a chemistry program around the cyclic guanidinium scaffoldusing a flexible synthetic route. Based on this practical syntheticroute, we have assembled structure-activity relationship (SAR) data. SeeFIG. 3. The chiral secondary alcohol in compounds 3 (marked with a *) isclearly important as oxidation to a ketone (see compounds 4a-b, 4f,4h-i) totally abolishes activity. Also, acetylation of this alcohol andthe imine nitrogen results in inactive compounds (see structure 5). SARfor the top aromatic ring indicates that bulky (CMe₃, compound 3e) orelectron-withdrawing (NO₂, compound 3d) substitution in thepara-position is dendrimental to activity. The relative activity in theseries 3f>3b>3c highlights the preference for a para-methoxysubstitutent. The ortho-nitro substituted analog 3h is equipotent to thedichloro-substituted lead 3a.

Example 6

Orexin Peptides and Agonists Prevent Cataplexy and Improve Wakefulness

Aspects of this example, including more detailed descriptions of theproduction of the CAG/orexin-transgenic mice, the cataplexy testing,cannulation (i.c.v.) and orexin administration and EEG/EMG recordingswere published in Mieda et al., PNAS USA Mar. 30, 2004; 101(13):4649-54.

We administered orexin-A, a nonselective agonist for orexin receptortype 1 (OX1R) and OX2R. The dose and route of administration chosen (3nmol per mouse i.c.v.) is comparable to that used previously to examinethe effects of orexin-A on behavior and metabolism in rodents, and iseffective in dose-response studies of sleep/wakefulness in wild-typemice, but produces no detectable effects in knockout mice lacking bothOX1R and OX2R genes.

We examined whether i.c.v. orexin-A administration suppressescataplectic arrests in orexin/ataxin-3-transgenic mice. By using arandomized crossover design, we administered vehicle and orexin-A to allmice during separate experimental sessions. When narcoleptic mice weretreated with vehicle alone, they exhibited variable frequencies ofarrests, as well as variable cumulative times spent in arrests duringthe first 3 h after injections. In contrast, when the same mice wereadministered orexin-A, the frequency of arrests and overall time spentin cataplexy were significantly reduced in each mouse during the same3-h time period, despite increases in observable wakeful activity. Thus,i.c.v. administration of orexin-A is sufficient to acutely suppressbehavioral arrests in this model of narcolepsy.

To quantify the effects of orexin-A administration on sleep/wake status,we recorded EEG/EMG in orexin/ataxin-3 mice, as well as wild-typecontrols. Because narcoleptic humans and animals exhibit sleepiness andcataplexy during their respective active phases, orexin-based therapiesare preferably administered to humans during the active phase.Nevertheless, some pharmacological effects of exogenous orexins havebeen reported to vary with time of day (Zeitgeber-dependent effects). Wetherefore examined the effects of orexin-A administrations in mice bothat the onset of the dark (active) phase as well as during the light(resting) phase, again by using a randomized crossover design.

Patterns of wakefulness, non-REM sleep, and REM sleep revealed a robustarousal effect of orexin-A in transgenic narcoleptic as well aswild-type mice. Central administration of 3 nmol of orexin-A strikinglyincreased wakefulness and suppressed both non-REM and REM sleep,regardless of genotype and time of administration. Interestingly,equivalent doses of orexin-A, administered during either the nocturnalor diurnal phases, produced arousal with greater effectiveness innarcoleptic mice compared with wild-type controls. Whereas bothwild-type and narcoleptic mice exhibited similar amounts of wakefulnessand non-REM sleep in a 3-h period after vehicle administrations,orexin-A induced significantly greater amounts of wakefulness inorexin/ataxin-3-transgenic mice than it did in wild-type mice. Theseincreases in wakefulness were essentially mirrored by significantlygreater suppressions of non-REM sleep in narcoleptic animals.

As expected, orexin/ataxin-3 mice exhibited significantly higher amountsof REM sleep than did wild-type controls during the dark phase underbaseline (vehicle-administered) conditions. Critically, orexin-Aeffectively suppressed this elevation of REM sleep in narcoleptic mice.Again, orexin-A was more effective at suppressing REM sleep innarcoleptic mice than in wild-type controls, regardless of the time ofadministration.

Unlike effects reported with amphetamine administrations in humans,rats, and mice, no immediate rebounds of sleep were observed afterdark-phase or light-phase orexin-A administrations. Narcoleptic animalsmaintained a statistically significant increase of cumulativewakefulness and a decrease of cumulative sleep, even after 24 h after anocturnal administration of orexin-A. When orexin-A was administereddiurnally, mice of both genotypes recovered sleep losses by the 24-hmark, but this recovery was gradual in nature.

The same protocols demonstrate that orexin agonists (e.g. Tables 1, 2;FIG. 3) similarly prevent cataplexy and improve wakefulness at 10 mg/kgadministered either orally in food or water, or i.p. Overall, theseresults demonstrate the feasibility of treating symptoms ofnarcolepsy-cataplexy by using pharmacological agonists of orexinreceptors.

Example 7

Orexin Agonists Abbreviate Emergence from General Anesthesia—Aspects ofthis Example were Published by Kelz et al. (PNAS, Jan. 29, 2008, vol.105, no. 4, p. 1309-1314)

Loss and Return of Righting Reflex. Induction and emergence fromisoflurane or sevoflurane were defined behaviorally as the respectiveloss and return of the righting reflex and were evaluated as follows.Wild-type C57BL/6J male mice aged 8-14 weeks (Jackson Laboratories).were placed in cylindrical gas-tight, controlled-environment chambersarrayed in parallel (35). After 90 min of habituation with 100% oxygeneach day on two successive days, anesthesia was induced with a Drägermodel 19.1 isoflurane or sevoflurane vaporizer by using 8-12 stepwiseincremental increases in the concentration of anesthetic gas dissolvedin 100% oxygen. Anesthetic gas concentrations were determined intriplicate during the last 2 min at each step (35). Initial volatileanesthetic concentrations were 0.44% isoflurane or 0.96% sevoflurane.After 15 min at each concentration to allow for equilibration of themouse with the anesthetic vapors, the concentration of volatileanesthetic was increased by 6±3% of the preceding value. Peak volatileanesthetic concentrations were 1.19% isoflurane or 1.52% sevoflurane. Atthe end of each 15-min interval, the cylindrical chambers were rotated180°. A mouse was considered to have lost the righting reflex if it didnot to turn itself prone onto all four limbs within 2 min After the lastmouse lost its righting reflex, volatile anesthetic concentration wasincreased one additional time before measurements of emergence time,which was defined as the duration that elapsed until each mouse regainedits righting reflex by turning prone onto all four feet. Mousetemperature was maintained between 36.6±0.6° C. by submerging thecontrolled environment chambers in a 37° C. water bath. In an effort tominimize both the number of mice used and the number of anestheticexposures, induction and emergence from anesthesia in orexin/ataxin-3mice and sibling controls were performed during the same experiment.

Pharmacologic Inhibition of Orexin Signaling and Righting ReflexStudies. To determine the effect of the orexin-1R antagonist,SB-334867-A (Tocris Bioscience), on induction of anesthesia, 24 C57BL/6Jmice were evaluated for both the concentration at which the rightingreflex was lost as well as a separate experiment to measure the latencyto loss of righting. Thirty minutes before determining isofluranesensitivity, one-third of the mice received an i.p. injection of vehicle(10% encapsin and 2% DMSO in sterile water), and the other two-thirdsreceived 5 mg/kg or 20 mg/kg of freshly prepared SB-334867-A dissolvedin vehicle (21) and administered i.p. at 20 ml/kg. All three groups ofmice were exposed to 1.25% isoflurane. Righting reflex was checked every15 s. To determine the dose-response curve for loss of righting,stepwise increases in isoflurane concentration were performed every 15min as described above. To eliminate the questions about the duration ofaction of SB-334867-A, emergence from isoflurane anesthesia in wild-typeC57BL/6J mice was studied as described below rather than after thetypical 3 h required to generate induction dose-response curves.C57BL/6J mice were anesthetized in controlled-environment chambers (35)with 1.25% isoflurane for 90 min. Individual mice were rapidly removed,injected with an i.p. bolus of either vehicle, 5 mg/kg, or SB-334867-A,20 mg/kg, doses known to reverse orexin-A-mediated behaviors (21, 22).All i.p. volumes were delivered at 20 ml/kg over 20-30 s. Immediatelyafter injection, mice were returned to the controlled-environmentchambers where they continued to breathe 1.25% isoflurane for anadditional 30 min before anesthetic gases were discontinued andemergence time_(RR) was recorded as described above

Results. While orexin-1R antagoinist SB-334867-A produced adose-dependent delay in emergence of wild type C57BL/6J mice exposed toisoflurane, and significantly delayed emergence at 20 mg/kg i.p.(F_(2,45)=8.80, P<0.001), the same protocols demonstrate that orexinagonists (e.g. Tables 1, 2, FIG. 3) produce a dose-dependentabbreviation in emergence of wild type C57BL/6J mice exposed toisoflurane, and significantly abbreviated emergence at 10 mg/kgadministered either orally in food or water, or i.p.

Example 8

OX2R selective agonists abbreviate recovery from jet lag. An establishedanimal model may be used to assess the effects of orexin agonists on jetlag. In one suitable protocol, the rhythms in rest/activity and bodytemperature are studied in male 6-week-old B6D2F1 mice having a radiotransmitter (Physio Tel, TA 10 TA-F20; Data Sciences, St. Paul, Minn.)implanted into the peritoneal cavity, which records locomotor activityand body temperature every 10 minutes. The mice are synchronized tostandard lighting conditions of LD12:12, with lights on from 6 a.m. to 6p.m., for 3 weeks and then undergo experimental jet lag produced by an8-hour advance of light/dark cycle. Control and treatment mice are thenmonitored for the time taken to recalibrate the animals' rest/activityand temperature rhythms to the new cycle. Use of this protocoldemonstrates that orexin agonists (e.g. Tables 1, 2, FIG. 3) abbreviatethe time required to reset circadian rhythms shifted by jet lag at 10mg/kg administered either orally in food or water, or i.p.

Example 9

OX2R Selective Agonists Reduce Diet-induced Obesity

To examine whether enhanced OX2R signaling causes resistance todiet-induced obesity, an OX2R selective agonist, [Ala11, D-Leu15]Orexin-B (Akanmu and Honma, 2005 Brain. Res. 1048.138-145.; Asahi etal., 2003 Bioorg. Med. Chem. Lett. 13.

111-113) was continuously infused in the lateral ventricles of wild-typemice for 14 days. The administration of the OX2R selective agonist (0.5nmol/d) suppressed weight gain on a high fat diet, without alteringweight homeostasis on a low fat diet. There was no effect of the OX2Rselective agonist for OX2R-deficient mice on a high fat diet (n=4,weight gain 3.33±0.61 g), verifying the selectivity of the agonist invivo. Following 14 days, the agonist-infused wild-type mice gainedsignificantly less fat mass than did the vehicle-injected mice on a highfat diet, and no effect was observed on a low fat diet. Administrationof the OX2R selective agonist reduced food intake only on a high fat butnot on low fat diet. After 14 days of OX2R agonist administration, weobserved reduced hypothalamic mRNA expression of orexigenic factors, NPYand AGRP on a high fat diet, compared to those on a low fat diet. Wedetected no significant change in LEPR, or other downstream transcriptsPOMC, SOCS3, or STAT3. The same experimental protocol demonstrates thatorexin agonists (e.g. Tables 1, 2, FIG. 3) similarly inhibitdiet-induced obesity at 10 mg/kg administered either orally in food orwater, or i.p.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method for enhanced wakefulness of a person subject determined tobe in need thereof and having narcolepsy or other neurologic conditionaccompanying daytime sleepiness, comprising the step of: (a) orallyadministering to the subject a type-2 orexin receptor (OX2R) agonistformula I:

in which: R₁ is substituted or unsubstituted alkyl; substituted orunsubstituted cycloalkyl; substituted or unsubstituted alkenyl;adamantyl; substituted or unsubstituted phenyl; a 5- or 6-memberedoptionally substituted saturated or unsaturated heterocyclic grouphaving from one to three heteroatoms selected from nitrogen, oxygen andsulfur; substituted or unsubstituted benzyl; a saturated or unsaturatedfused ring carbocyclic group having from 8 to 10 ring atoms; or CH₂XR₅,where X is oxygen, sulfur, —NH— or —CH₂— and R₅ is substituted orunsubstituted alkyl; substituted or unsubstituted cycloalkyl;substituted or unsubstituted phenyl; substituted or unsubstitutedbenzyl; 2-carbamide-indolyl; or a 5 to 9-membered optionally substitutedsaturated or unsaturated heterocyclic group having from one to threeheteroatoms selected from nitrogen, oxygen and sulfur; R₂ is substitutedor unsubstituted alkyl; substituted or unsubstituted cycloalkyl;substituted or unsubstituted alkenyl; substituted or unsubstitutedalkoxyalkyl; a 5- or 6-membered optionally substituted saturated orunsaturated heterocyclic group having from one to three heteroatomsselected from nitrogen, oxygen and sulfur; —(CH₂)_(n)R₃, where R₃ is (i)a 5- to 9-membered optionally substituted saturated or unsaturatedheterocyclic group having from one to three heteroatoms selected fromnitrogen, oxygen and sulfur; (ii) —NR₆R₇ where R₆ and R₇ areindependently selected from hydrogen, methyl, ethyl and benzyl; or (iii)COOR₈ where R₈ is alkyl; and n is 2 or 3; substituted or unsubstitutedphenyl; substituted or unsubstituted benzyl; a saturated or unsaturatedfused ring carbocyclic group having from 8 to 10 ring atoms; ormethylene-1-naphthyl; and R₄ is hydrogen; (CH₂)_(m)COOR₁₅ where R₁₅ isalkyl or substituted alkyl; and m is 0, 1 or 2; CONR₁₆R₁₇, where R₁₆ andR₁₇ are independently (i) hydrogen; (ii) alkyl or substituted alkyl;(iii) cycloalkyl; (iv) alkoxyalkyl; (v) a 5- to 10-membered optionallysubstituted saturated or unsaturated heterocyclic group having from oneto three heteroatoms selected from nitrogen, oxygen and sulfur; (vi) asaturated or unsaturated fused ring carbocyclic group having from 8 to10 ring atoms; (vii) substituted or unsubstituted phenyl; (viii)(CH₂)_(p)R₁₈ where R₁₈ is a 5- or 6-membered optionally substitutedsaturated or unsaturated heterocyclic group having from one to threeheteroatoms selected from nitrogen, oxygen and sulfur, and p is 1, 2 or3; (ix) optionally substituted benzyl; or (x) an aralkyl groupcomprising a chain of from 1 to 4 methylene groups substituted by one ortwo phenyl groups; C₁-C₄ alkoxy; optionally substituted phenoxy;SO₂NR₁₉R₂₀ where R₁₉ and R₂₀ are independently hydrogen, optionallysubstituted alkyl or phenyl; NR₂₁R₂₂, wherein R₂₁ and R₂₂ areindependently hydrogen, alkyl, optionally substituted phenyl oroptionally substituted benzyl; COR₂₃ where R₂₃ is alkyl or is NR₂₁R₂₂,COOR₂₄ where R₂₄ is hydrogen, alkyl, or benzyl; or SO₂R₂₅ where R₂₅ isalkyl or NR₂₁R₂₂; or a pharmaceutically acceptable salt thereof.
 2. Amethod according to claim 1 in which R₁ is C₁-C₆ alkyl, optionallysubstituted with one or more groups selected from halo, hydroxy, cyano,nitro, COOH and COOCH₃; C₂-C₆ alkenyl, optionally substituted with oneor more groups selected from halo, hydroxy, cyano, nitro, COOH andCOOCH₃; C₃-C₆ cycloalkyl, optionally substituted with one or more groupsselected from halo, hydroxy, cyano, nitro, COOH and COOCH₃; adamantyl;optionally substituted phenyl in which the substituents are selectedfrom mono- and dihalo, mono- and di-(C₁-C₄ alkoxy), mono- and di-(C₁-C₄alkylthio), C₁-C₂ alkyleneoxy; C₁-C₂ alkylenedioxy, mono- and di-(C₁-C₄alkyl), mono- and di-(trifluoromethyl), mono- and di-(C₁-C₄hydroxyalkyl), mono- and di-(C₁-C₄ alkoxyalkyl), mono- and di-hydroxy,mono- and di-cyano, mono- and di-(COCH₃), and mono- and di-NHR₂₆,wherein R₂₆ is COCH₃, SO₂CH₃, SO₂C₆H₅, COOR′ or CONR′R″; and wherein R′and R″ are independently hydrogen or C₁-C₄ alkyl; a 5- or 6-memberedsaturated or unsaturated heterocyclic group having from one to threeheteroatoms selected from nitrogen, oxygen and sulfur, optionallysubstituted by from 1 to 3 groups independently selected from halo,C₁-C₄ alkyl, C₁-C₄ alkoxy, oxo and hydroxy; optionally substitutedbenzyl in which the substituents are selected from mono- and dihalo,mono- and di-(C₁-C₄ alkoxy), mono- and di-(C₁-C₄ alkylthio), C₁-C₂alkyleneoxy; C₁-C₂ alkylenedioxy, mono- and di-(C₁-C₄ alkyl), mono- anddi-(trifluoromethyl), mono- and di-(C₁-C₄ hydroxyalkyl), mono- anddi-(C₁-C₄ alkoxyalkyl), mono- and di-hydroxy, mono- and di-cyano, mono-and di-(COCH₃), and mono- and di-NHR₂₆; naphthyl; or CH₂XR₅, where X isoxygen, sulfur, —NH— or —CH₂— and R₅ is selected from (i) C₁-C₆ alkyl;(ii) C₃-C₆ cycloalkyl; (iii) optionally substituted phenyl in which thesubstituents are selected from mono- and di-(C₁-C₄ alkyl), mono- anddihalo, mono and di-(C₁-C₄ alkoxy), C₁-C₂ alkyleneoxy, C₁-C₂alkylenedioxy, mono- and di-(trifluoromethyl), nitro, hydroxy, mono- anddi-(C₁-C₄ hydroxyalkyl), mono- and di-(C₁-C₄ alkoxyalkyl), mono- anddi-hydroxy, mono- and dicyano, mono- and di-(COCH₃) and mono- anddi-NHR₂₆; (iv) benzyl; (v) 2-carbamide-indolyl; or (vi) a 5- to9-membered saturated or unsaturated heterocyclic group having from oneto three heteroatoms selected from nitrogen, oxygen and sulfur,optionally substituted by from 1 to 3 groups independently selected fromhalo, C₁-C₄ alkyl, C₁-C₄ alkoxy, oxo and hydroxy; R₂ is C₁-C₁₂ alkyl,optionally substituted with one or more groups selected from halo,hydroxy, cyano, nitro, COOH and COOCH₃; C₂-C₈ cycloalkyl, optionallysubstituted with one or more groups selected from halo, hydroxy, cyano,nitro, COOH and COOCH₃; C₂-C₁₂ alkenyl, optionally substituted with oneor more groups selected from halo, hydroxy, cyano, nitro, COOH andCOOCH₃; C₂-C₁₂ alkoxyalkyl; a 5- or 6-membered saturated or unsaturatedheterocyclic group having from one to three heteroatoms selected fromnitrogen, oxygen and sulfur, optionally substituted by from 1 to 3groups independently selected from halo, C₁-C₄ alkyl, C₁-C₄alkoxy, oxo,and hydroxy; (CH₂)nR₃, where R₃ is (i) a 5- to 9-membered saturated orunsaturated heterocyclic group having from one to three heteroatomsselected from nitrogen, oxygen and sulfur, optionally substituted byfrom 1 to 3 groups independently selected from halo, C₁-C₄ alkyl, C₁-C₄alkoxy, oxo, and hydroxy; (ii) —NR₆R₇ where R₆ and R₇ are independentlyselected from hydrogen, methyl, ethyl and benzyl; or (iii) COOR₈ whereR₈ is C₁-C₄ alkyl; and n is 2 or 3; optionally substituted phenyl, wherethe substituents are independently selected from mono- and di-(C₁-C₄alkyl); mono- and dihalo; mono-, di-, and tri-(C₁-C₄ alkoxy); mono- anddicyano; nitro; methylsulfonyl; mono-, di-, and tri-(trifluoromethyl);C₁-C₂ alkyleneoxy; C₁-C₂ alkylenedioxy; —(CH₂)_(q)COOR₉ where R₉ isC₁-C₄ alkyl; or —(CH₂)_(q)N R₁₀R₁₁ where R₁₀ and R₁₁ are independentlyselected from hydrogen, C₁-C₄ alkyl, COR₁₂ where R₁₂ is C₁-C₄ alkyl,SO₂R₁₃ where R₁₃ is C₁-C₄ alkyl, or COOR₁₄ where R₁₄ is C₁-C₄ alkyl andCONR′R″; and q is an integer from 1 to 4; optionally substituted benzyl,where the substituents are selected from mono-, di-, and tri-(C₁-C₄)alkyl; mono-, di-, and tri-halo; mono-, di-, and tri-(C₁-C₄ alkoxy);mono-, di-, and tri-(C₁-C₄ alkylthio); mono- and di-cyano; nitro;methylsulfonyl; mono-, di-, and tri-(trifluoromethyl); mono-, di-, andtri-COOR′; mono-, di-, and triCONR′R″; mono-, di-, and tri-NR₂₇R₂₈ whereR₂₇ and R₂₈ are independently selected from hydrogen, C1-C4 alkyl,acetyl, and methylsulfonyl; C₁-C₂ alkyleneoxy; C₁-C₂ alkylenedioxy; andmono-, di-, and tri-(C₁-C₆ alkoxyalkyl); a saturated or unsaturatedfused ring carbocyclic group having from 8 to 10 ring atoms; ormethylene-1- or 2-naphthyl; and R₄ is hydrogen; (CH₂)_(m)COOR′ where mis 0, 1 or 2; CONR₁₆R₁₇ where R₁₆ and R₁₇ are independently (i)hydrogen; (ii) C₁-C₅ alkyl optionally substituted with one or moregroups selected from halo, hydroxy, cyano, nitro, COOH and COOCH₃; (iii)C₃-C₆ cycloalkyl; (iv) C₂-C₈ alkoxyalkyl; (v) a 5- to 10-memberedsaturated or unsaturated heterocyclic group having from one to threeheteroatoms selected from nitrogen, oxygen and sulfur, optionallysubstituted by from 1 to 3 groups independently selected from halo,hydroxy, oxo, C₁-C₄ alkyl, C₁-C₄ alkoxy, COCH₃, COOR′, and NR₂₉R₃₀ whereR₂₉ and R₃₀ are independently selected from hydrogen, C₁-C₄ alkyl,acetyl, or methylsulfonyl; (vi) a saturated or unsaturated fused ringcarbocyclic group having from 8 to 10 ring atoms; (vii) (CH₂)_(p)R₁₈where R₁₈ is a 5- or 6-membered saturated or unsaturated heterocyclicgroup having from 1 to 3 heteroatoms selected from nitrogen, oxygen andsulfur, optionally substituted by from 1 to 3 groups independentlyselected from halo, hydroxyl, C₁-C₄ alkyl, C₁-C₄ alkoxy and oxo, and pis 1, 2 or 3; (viii) phenyl optionally substituted by one or more groupsindependently selected from mono-, di-, and tri-halo, mono-, di-, andtrihydroxy, mono-, di-, and tri-(C₁-C₄ alkyl), C₁-C₂ alkyleneoxy, C₁-C₂alkylenedioxy, COOR′, and NR₂₉R₃₀ where R₂₉ and R₃₀ are independentlyselected from hydrogen, C₁-C₄ alkyl, acetyl, and methylsulfonyl; (ix)optionally substituted benzyl where the substituents are selected frommono-, di-, and tri-(C₁-C₄ alkyl); mono-, di-, and trihalo; mono-, di-,and tri-(C₁-C₄ alkoxy); nitro; methylsulfonyl; mono-, di-, andtri-(trifluoromethyl); NR′R′″; C₁-C₂ alkyleneoxy; C₁-C₂ alkylenedioxy;(x) an aralkyl group comprising a chain of from 1 to 4 methylene groupssubstituted by one or two phenyl groups; C₁-C₄ alkoxy; optionallysubstituted phenoxy, where the substituents are independently selectedfrom mono- and di-(C₁-C₄) alkyl; mono- and dihalo; mono-, di-, andtri-(C₁-C₄ alkoxy); cyano; nitro; methylsulfonyl; mono-, di-, andtri-trifluoromethyl; C₁-C₄ alkyleneoxy; C₁-C₂ alkylenedioxy;—(CH₂)_(r)COOR₉ where R₉ is C₁-C₄ alkyl; or (CH₂)_(r)NR₃₀R₃₁, where R₃₀and R₃₁ are independently hydrogen, C₁-C₄ alkyl, COR₃₂ where R₃₂ isC₁-C₄ alkyl, SO₂R₃₃ where R₃₃ is C₁-C₄ alkyl, or COOR₃₄ where R₃₄ isC₁-C₄ alkyl; SO₂NR₃₅R₃₆ where R₃₅ and R₃₆ are independently hydrogen,C₁-C₄ alkyl or phenyl; or NR₃₇R₃₈ where R₃₇ and R₃₈ are independentlyhydrogen; C₁-C₄ alkyl; phenyl; COR₃₉ where R₃₉ is C₁-C₄ alkyl; or SO₂R₄₀where R₄₀ is hydrogen or C₁-C₄ alkyl.
 3. A method according to claim 1in which R₁ is phenyl, substituted phenyl, a 5- or 6-membered saturatedor unsaturated heterocyclic group having from one to three heteroatomsselected from nitrogen, oxygen and sulfur optionally substituted by from1 to 3 groups independently selected from halo, alkyl, alkoxy, oxo orhydroxy, or CH₂XR; R₂ is benzyl, substituted benzyl, C₂-C₄ alkyl, or—(CH₂)₂R₃, where R₃ is a 5- to 6-membered saturated or unsaturatedheterocyclic group having from one to three heteroatoms selected fromnitrogen, oxygen and sulfur; and R₄ is hydrogen.
 4. A method accordingto claim 1 in which R₁ is optionally substituted phenyl.
 5. A methodaccording to claim 1 in which R₁ is mono- or dihalophenyl.
 6. A methodaccording to claim 1 in which R₁ is 3,4-dichlorophenyl.
 7. A methodaccording to claim 1 in which R₂ is benzyl or substituted benzyl.
 8. Amethod according to claim 2 in which R₂ is benzyl or substituted benzyl.9. A method according to claim 2 in which R₂ is benzyl substituted by aC₁-C₄ alkyl group, a C₁-C₄ alkoxy group, or a halogen.
 10. A methodaccording to claim 2 in which R₂ is 4-methylbenzyl, 4-methoxybenzyl, or4-fluorobenzyl.
 11. A method according to claim 1 in which R₄ ishydrogen.
 12. The method of claim 1, further comprising detecting aresultant enhanced wakefulness.
 13. The method of claim 1 comprisingadministering to the subject the OX2R agonist regularly and chronically.14. A method for enhanced wakefulness of a person subject determined tobe in need thereof and having narcolepsy or other neurologic conditionaccompanying daytime sleepiness, comprising the step of: (a) orallyadministering to the subject a type-2 orexin receptor (OX2R) agonist offormula I:

in which: R₁ is substituted or unsubstituted alkyl; substituted orunsubstituted cycloalkyl; substituted or unsubstituted alkenyl;adamantyl; substituted or unsubstituted phenyl; a 5- or 6-memberedoptionally substituted saturated or unsaturated heterocyclic grouphaving from one to three heteroatoms selected from nitrogen, oxygen andsulfur; substituted or unsubstituted benzyl; a saturated or unsaturatedfused ring carbocyclic group having from 8 to 10 ring atoms; or CH₂XR₅,where X is oxygen,sulfur,—NH— or —CH₂— and R₅ is substituted orunsubstituted alkyl; substituted or unsubstituted cycloalkyl;substituted or unsubstituted phenyl; substituted or unsubstitutedbenzyl; 2-carbamide-indolyl; or a 5 to 9-membered optionally substitutedsaturated or unsaturated heterocyclic group having from one to threeheteroatoms selected from nitrogen, oxygen and sulfur; R₂ is substitutedor unsubstituted alkyl; substituted or unsubstituted cycloalkyl;substituted or unsubstituted alkenyl; substituted or unsubstitutedalkoxyalkyl; a 5-or 6-membered optionally substituted saturated orunsaturated heterocyclic group having from one to three heteroatomsselected from nitrogen, oxygen and sulfur; —(CH₂)_(n) R₃, where R₃ is(i) a 5-to 9-membered optionally substituted saturated or unsaturatedheterocyclic group having from one to three heteroatoms selected fromnitrogen, oxygen and sulfur; (ii) —NR₆R₇ where R₆ and R₇ areindependently selected from hydrogen, methyl, ethyl and benzyl; or (iii)COOR₈ where R₈ is alkyl; and n is 2 or 3; substituted or unsubstitutedphenyl; substituted or unsubstituted benzyl; a saturated or unsaturatedfused ring carbocyclic group having from 8 to 10 ring atoms; ormethylene-1-naphthyl; and R₄ is hydrogen; (CH₂)_(m)COOR₁₅ where R₁₅ isalkyl or substituted alkyl; and m is 0, 1 or 2; CONR₁₆R₁₇, where R₁₆ andR₁₇ are independently (i) hydrogen; (ii) alkyl or substituted alkyl;(iii) cycloalkyl; (iv) alkoxyalkyl; (v) a 5- to 10-membered optionallysubstituted saturated or unsaturated heterocyclic group having from oneto three heteroatoms selected from nitrogen, oxygen and sulfur; (vi) asaturated or unsaturated fused ring carbocyclic group having from 8 to10 ring atoms; (vii) substituted or unsubstituted phenyl; (viii)(CH₂)_(p)R₁₈ where R₁₈ is a 5-or 6-membered optionally substitutedsaturated or unsaturated heterocyclic group having from one to threeheteroatoms selected from nitrogen, oxygen and sulfur, and p is 1, 2 or3; (ix) optionally substituted benzyl; or (x) an aralkyl groupcomprising a chain of from 1 to 4 methylene groups substituted by one ortwo phenyl groups; C₁-C₄ alkoxy; optionally substituted phenoxy;SO₂NR₁₉R₂₀ where R₁₉ and R₂₀ are independently hydrogen, optionallysubstituted alkyl or phenyl; NR₂₁R₂₂, wherein R₂₁ and R₂₂ areindependently hydrogen, alkyl, optionally substituted phenyl oroptionally substituted benzyl; COR₂₃ where R₂₃ is alkyl or is NR₂₁R₂₂;COOR₂₄ where R₂₄ is hydrogen, alkyl, or benzyl; or SO₂R₂₅ where R₂₅ isalkyl or NR₂₁R₂₂; or a pharmaceutically acceptable salt thereof, andfurther comprising administering to the subject the OX2R agonist inconjunction with an effective amount of a different compound whichpromotes wakefulness.
 15. The method of claim 1 comprising administeringto the subject the OX2R agonist in unit dosage form, and comprising theantecedent step of removing a capsule or tablet comprising the unitdosage form from a multipack adapted for sequential use.
 16. A methodfor promoting enhanced wakefulness of a person subject determined to bein need thereof and having narcolepsy or other neurologic conditionaccompanying daytime sleepiness, comprising the step of: (a) orallyadministering to the subject a type-2 orexin receptor (OX2R) agonist offormula:

or a pharmaceutically acceptable salt thereof.